Bottom Line:
Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes.This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise.Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota - Twin Cities, Minneapolis, Minnesota, United States of America.

ABSTRACTCell-to-cell gene expression noise is thought to be an important mechanism for generating phenotypic diversity. Furthermore, telomeric regions are major sites for gene amplification, which is thought to drive genetic diversity. Here we found that individual subtelomeric TLO genes exhibit increased variation in transcript and protein levels at both the cell-to-cell level as well as at the population-level. The cell-to-cell variation, termed Telomere-Adjacent Gene Expression Noise (TAGEN) was largely intrinsic noise and was dependent upon genome position: noise was reduced when a TLO gene was expressed at an ectopic internal locus and noise was elevated when a non-telomeric gene was expressed at a telomere-adjacent locus. This position-dependent TAGEN also was dependent on Sir2p, an NAD+-dependent histone deacetylase. Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes. This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise. Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

pgen-1004436-g004: Subtelomeric TLOs exhibit cell-to-cell variance.(A) Tloα34, Tloα10, and Tloα12 tagged with GFP at the C-terminus were imaged to determine nuclear signal intensity of single cells. (B) Mean nuclear abundance of single cells GFP tagged at five Tlos was quantified using images as collected in (A). Mean GFP abundance of Tlos was similarly variable to the chromosome internal Tlo, Tloα34. However, variation of GFP abundance among single cells in a single replicate was greater for subtelomeric Tlos than Tloα34. At least four replicates were performed for each strain. (C) Flow cytometry profiles of Nup49, Tloα10, and Tloα12 tagged with GFP indicate the expression noise of cell within each population. An overlay of at least four experiments is shown.

Mentions:
Strikingly, the fluorescence signal for subtelomeric Tlo genes varied dramatically from cell-to-cell, ranging from very bright cells to cells with no obvious signal (Fig. 4A, B). The level of population-to-population variation was also higher for subtelomeric Tlo genes, consistent with the detection of expression plasticity at the population level (Fig. 1). Growth under stress conditions (5 mM H2O2 or cell wall stress) also resulted in high levels of Tloα12 cell-to-cell variation (Fig. S2; p<0.001; significance determined using a bootstrap procedure that compared the measured ratio of CVNup49-GFP/CVTloα12-GFP against the critical value obtained from 10,000 simulated datasets that randomized the background of measured cells). Consistent with the RNA-seq results, the non-telomeric Tloα34-GFP gene, exhibited minimal cell-to-cell and population-to-population variation (Fig. 4A, B).

pgen-1004436-g004: Subtelomeric TLOs exhibit cell-to-cell variance.(A) Tloα34, Tloα10, and Tloα12 tagged with GFP at the C-terminus were imaged to determine nuclear signal intensity of single cells. (B) Mean nuclear abundance of single cells GFP tagged at five Tlos was quantified using images as collected in (A). Mean GFP abundance of Tlos was similarly variable to the chromosome internal Tlo, Tloα34. However, variation of GFP abundance among single cells in a single replicate was greater for subtelomeric Tlos than Tloα34. At least four replicates were performed for each strain. (C) Flow cytometry profiles of Nup49, Tloα10, and Tloα12 tagged with GFP indicate the expression noise of cell within each population. An overlay of at least four experiments is shown.

Mentions:
Strikingly, the fluorescence signal for subtelomeric Tlo genes varied dramatically from cell-to-cell, ranging from very bright cells to cells with no obvious signal (Fig. 4A, B). The level of population-to-population variation was also higher for subtelomeric Tlo genes, consistent with the detection of expression plasticity at the population level (Fig. 1). Growth under stress conditions (5 mM H2O2 or cell wall stress) also resulted in high levels of Tloα12 cell-to-cell variation (Fig. S2; p<0.001; significance determined using a bootstrap procedure that compared the measured ratio of CVNup49-GFP/CVTloα12-GFP against the critical value obtained from 10,000 simulated datasets that randomized the background of measured cells). Consistent with the RNA-seq results, the non-telomeric Tloα34-GFP gene, exhibited minimal cell-to-cell and population-to-population variation (Fig. 4A, B).

Bottom Line:
Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes.This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise.Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

Affiliation:
Department of Genetics, Cell Biology and Development, University of Minnesota - Twin Cities, Minneapolis, Minnesota, United States of America.

ABSTRACTCell-to-cell gene expression noise is thought to be an important mechanism for generating phenotypic diversity. Furthermore, telomeric regions are major sites for gene amplification, which is thought to drive genetic diversity. Here we found that individual subtelomeric TLO genes exhibit increased variation in transcript and protein levels at both the cell-to-cell level as well as at the population-level. The cell-to-cell variation, termed Telomere-Adjacent Gene Expression Noise (TAGEN) was largely intrinsic noise and was dependent upon genome position: noise was reduced when a TLO gene was expressed at an ectopic internal locus and noise was elevated when a non-telomeric gene was expressed at a telomere-adjacent locus. This position-dependent TAGEN also was dependent on Sir2p, an NAD+-dependent histone deacetylase. Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes. This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise. Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.